Nucleic acid chemistries and analyses have risen to a place of prominence in such diverse areas as biological research, medicine, agriculture, and even forensic science. A key need in the use of nucleic acids in all of these areas is the ability to manipulate them on a macroscopic scale by localizing particular nucleic acid species (or groups of species) at a known location, such as in an array on a substrate. In order to immobilize nucleic acids on support materials, a wide range of methods have been devised, which can be loosely classified by the stability of the bond. A covalent immobilization, i.e. an immobilization in which the nucleic acid is linked to molecular structures of the support material by covalent bonds, is typically irreversible without risking the degradation of the immobilized nucleic acid. In contrast, complexing reactions, or reactions between two partners of a binding system which specifically recognize each other, may be used. These reactions may be reversible or, for practical purposes, irreversible over the timescale of a particular experiment. Whether a binding system for immobilizing nucleic acids is to be denoted reversible or irreversible depends ultimately on the position of the equilibrium between bound and free nucleic acids. An example of a binding system to be regarded as practically irreversible is the complexing of biotin by avidin (or streptavidin, or their various engineered equivalent proteins,) with a binding constant of Ka˜2.5×1013 (M−1) (Chilkoti, A.; Stayton, P. A.; J. Am. Chem. Soc. 117, 10622-10628 (1995); Greene M.; Advances in Protein Chemistry; 85-132 (1975)). This system has been widely used for immobilizing nucleic acids and other biomolecules on support materials.
WO 86/07387 describes a different example of reversible binding of nucleic acids to surfaces. As described here, nucleic acid binding sequences are immobilized on support materials by means of complexing agents, for example with the aid of antibody/antigen pairs.
Another example of a reversible pairing system which has been used are sets of natural-nucleotide oligomers, which specifically hybridize to provide duplexing “tags” for immobilization. In contrast to biotin-streptavidin, nucleic acid oligomers have an informatic dimensionality in pair formation: there are a multiplicity of specific binding sets which may be devised, characterized by the sequence of monomers (nucleotides) which specifically pair only with complementary sequences. Unlike multi-hapten-lectin systems, or multi-antibody-antigen systems, such sets of nucleic acid tags have fairly uniform chemical and thermodynamic characteristics, which facilitates their use in multiplex reactions.
EP 0 305 145 describes, for example, the use of homopolynucleotide tails and their specific pairing with complementary homopolynucleotide oligomers for immobilizing target-specific oligonucleotides. On the other hand, JP 03 151 900 describes the use of specific nucleic acid sequences for immobilizing target-specific nucleic acids. It is characteristic of such systems that the oligonucleotide to be immobilized is composed of two parts: one oligomeric part complementary to the oligonucleotide immobilized on the surface of the support, and a second oligomeric part specific for interaction with components of the sample (e.g., complementary to a sample nucleic acid.) Similar systems are also described in WO 93/13225, WO 93/13223, WO 93/25563, U.S. Pat. No. 5,763,175, WO 97/32999, WO 00/58516 and in WO 00/60124.
The disadvantage of the methods described above is that the sequence used for the immobilization can potentially hybridize with the sequence to be immobilized, forming intramolecular secondary structures, may hybridize with another sequence to be immobilized, forming intermolecular secondary structures, or may hybridize with nucleic acids from the sample. The risk of such an unwanted or interfering interaction increases with the length of the nucleic acid(s) to be immobilized, as well as with the complexity of a sample (e.g., the possibility of contaminating nucleic acids from unknown organisms.)
Another disadvantage of the use of natural nucleic acids for immobilization is that the stability of duplexes of natural nucleic acids does not increase linearly in proportion to length (number of nucleotides in the sequence) over a large range, but rather approaches a limit which depends only on the relative percentage of CG to AT base pairs (“CG content”). Binding systems having a duplex stability exceeding the natural limit cannot be prepared using natural nucleic acids. This limitation is also problematic when applying various stringency conditions to the nucleic acid at its immobilized location: the immobilizing nucleic acid tags will also be subjected to the same stringency conditions (i.e., chaotropic agents, thermal conditions, or electrostatic forces), and may dissociate. See G. Michael Blackburn and Michael J. Gait, eds. Nucleic Acids in Chemistry and Biology, 2nd ed., 1996, Oxford University Press, New York.
Achieving a fine differentiation in stringency differentiation between immobilization tag interactions (which must remain hybridized) and the target-specific interactions (which are often discriminated at the single base pair mismatch level) is often difficult, especially under clinical-type conditions when the method must be particularly robust and consistent.
Another significant economic and time disadvantage of using natural nucleic acids as immobilization agents is that a certain minimum sequence length is required to reach a practical level of stability and selectivity of the immobilization. It is to typical to use 20-mers in order to achieve sufficient binding specificity. This results in the entire nucleic acid strand (composed of the sequence for recognizing the sample and the sequence for immobilization) becoming relatively long. The use very long sequences can be disadvantageous for several reasons. First, the use of long nucleic acid sequences increases the likelihood of secondary structure formation intramolecularly, and also increases the likelihood of transient or stable hybridization between multiple strands in solution.
Active electronic array devices have been described for the electrophoretic transport and manipulation of nucleic acids, see U.S. Pat. Nos. 6,245,508; 6,225,059; 6,051,380; and 6,017,696, the text of each of which is hereby incorporated by reference in their entirety. When manipulating nucleic acids on active electronic arrays, the use of shorter sequences is preferred. Electro-kinetic addressing and movement on the electronic chip array work particularly well with relatively short nucleic acids because of the better electrophoretic mobility of the smaller molecules. Thus, shorter sequences for use as immobilization complexing agents have increased utility in the context of active electronic arrays.
Another disadvantage of using natural systems for the immobilization of nucleic acids is that such systems can be easily degraded or destroyed during their use. In particular, degradation by enzymatic components of the sample, or even contaminating DNAses and RNAses from laboratory workers' fingertips, is a concern. Degradation or fragmentation by hydrolysis of the nucleic acids used for immobilization, in particular by enzymes such as, restriction enzymes, exonucleases or endonucleases, in not uncommon when a nucleic acid oligomer is allowed to sit at room temperature for a few days. Thus, the use of complexing agents for immobilization which are not subject to degradation or modification by naturally occurring enzymes is desirable.
In the course of the last few decades, a plurality of technologies have been developed to take advantage of the diverse natural variety of enzymes to modify nucleic acids. Restriction endonuclease reactions specifically cleave nucleic acids at defined sequence sites, and nucleases or other enzymes can be utilized to degrade or modify nucleic acids at either termini. In addition, polymerases and terminal transferases can be utilized to build nucleic acid oligomers from nucleotides. Ligase enzymes may also be utilized to connect, or ligate, different nucleic acid strands with one another, in single-strand template dependant, blunt-end double-stranded, or single-strand template independent manners. These enzymatic tools have become a mainstay of analytical biochemistry, and are necessary for almost any molecular biology research at some point. For instance, polymerases are commonly used for carrying out nucleic acid amplification reactions and sequencing reactions, which are both necessary components of the production of proteins of interest in research.
In most cases, purification steps or physical separation steps are required for handling at each stage of the enzymatic manipulations of nucleic acids. Nucleic acids are usually purified by precipitation, electrophoretic separation, or chromatographic steps. For isolation and immobilization purposes, nucleic acids are often modified with an affinity tag, like biotin. These biotin-modified nucleic acids can be bound stably and irreversibly to solid phases via macromolecular biotin-streptavidin complexes (e.g. DE 40 011 54 and EP 0 063 879). The very large complex obtained can be separated again only by rather harsh chemical conditions. Although a commonly used complex, this provides only one immobilization interaction tool: multiplex reactions with specific localization of the products cannot be done with a biotin-streptavidin affinity system alone. Alternatively, A is possible to elaborately incorporate polyhistidine modifications, or steroids or haptens, such as digoxigenin, into a nucleic acid in order to make separation possible by fixing to binding partners corresponding to the modifications. However, these systems only allow separation under diverse conditions (nickel chromatography, vs antibody binding,) and thus do not overcome the limitations of the biotin-avidin interaction for multiplex reactions.
Chemical conjugation of one nucleic acid to another nucleic acid in solution requires that one nucleic acid is provided with a modification which can react with the modification of the other nucleic acid by forming a stable bond. Often, finished, pre-synthesized nucleic acids are conjugated with other nucleic acids and analogs by utilizing the ability of nucleic acids to form complementary pairs with themselves, or by the pairing of the nucleic acids with a nucleic acid template for ligation. The pairing leads to an association or pre-organization of the parts to be conjugated, which supports or else makes thermodynamically favorable the subsequent conjugation reaction. For example, Nucleic Acids Research 16(9), 3671-3691 (1988) describes the conjugation of thiol-modified nucleic acids. This option allows, when treated with atmospheric oxygen, the formation of both homodimers and heterodimers, and is therefore only provisionally suitable for linking together two different nucleic acids. The template-supported conjugation of aldehyde-modified nucleic acids with amine-modified nucleic acids is described in J. Am. Chem. Soc. 114, 9197-9198, (1992). Another template-supported photochemical conjugation is described in Nucleic Acids Research 26(13), 3300-3304, (1998). One of the few reactions described without support by self-binding or template binding is the reaction of phosphorothioates with α-haloacetylene (Gryaznov S M, J. Am. Chem. Soc. 115, 3808-3809, (1993)). WO 01/07657 describes the linkage of RNA building blocks at the 3′ end of an RNA oligonucleotide by oxidation with periodate to give the dialdehyde and subsequent reaction with a nitrogen nucleophile to give a cyclic product. The nitrogen nucleophiles used may be amines, hydrazines, hydrazides, semicarbazides or thiosemicarbazides. The product formed initially can be stabilized by reduction with NaCNBH3.
Since synthetic binding systems, such as pyranosyl-RNA (pRNA) or pyranosyl-DNA (pDNA) binding systems, are, by design, not sterically capable of pairing with nucleic acids, these previously described methods are not applicable to conjugating to nucleic acids with synthetic binding units. However, the solid-phase tandem synthesis of synthetic binding unit/nucleic acid conjugates by phosphoramidite chemistry is not desirable for circumstances in which a nucleic acid is readily available for conjugation (e.g., a bacterial plasmid preparation.) Likewise, if the nucleic acid to be conjugated is longer than about 20 nucleotides, the size of the conjugate (nucleic acid +6-15 pRNA residues) approaches and eventually surpasses the efficient synthesis limit of the solid-phase chemistry. There is therefore a need to find methods and conditions which make it possible to conjugate finished, pre-synthesized nucleic acids with synthetic binding systems.